The arising of microarray technique since the end of last century has speeded the progress of Human Genome Project as well as many other research projects requiring high-through put analysis. Fluorescence microarray scanner has then become the most recently and successfully developed fluorescence detection instrument for the purpose of microarray chip analysis. But there appear two problems which affect its application in quantitative microarray analysis. For the individual scanners, the laser source inevitably fluctuates or even declines during operation and the confocal plane may also shift due to malfunctioned optical system, scanning accuracy also differs after long-term usage, which may cause error readings. For the instruments from different manufacturers, they differ in design and manufactory of light source, light path as well as the detection system, which results in incomparable readings. As all the other fluorescence detection instruments for quantitative analysis require routine calibration, fluorescence microarray scanner also needs to be calibrated to assure best performance. Thus, a calibration method for routine maintenance and data comparison between different instruments is inevitable. Fluorescent standard solutions have been used for calibration of fluorescence spectrometers and plate readers. However, they are not suitable for fluorescence microarray scanners which employ glass slide as detection targets. The object used as the calibration tool has to be easy to use, compatible to commonly used fluorescence channels in fluorescence microarray scanners, and most important, stable under long-term laser excitation.
At present, commonly used materials and means for calibration of fluorescence microarray scanners are as follows:
1. Organic fluorescent dyes as calibration material:
Gene Pharm Co. provides the Dilution Series Slide (DS3001) by directly printed Cy3, Cy5 dyes on the glass substrates to evaluate the stability of the laser scanner. The 20 serial dilutions cover several orders of concentration magnitude. Full Moon Biosystems produces similar calibration slide—FMB Microarray Scanner Calibration Slides. The slides are packaged in a vacuum-sealed bag and suggested to keep away from light at low temperature under dry condition.
Organic Fluorescence dyes possess poor photostability and will photo-bleach after exposure to light. The fluorescent intensity of the dyes (calibration dot) and thus shelf life of this kind of calibration slide are inevitable decreased after used in a relatively short time.
2. Organic Fluorescence Dyes Doped Polymers as Calibration Material:
Starna® fluorescence standards supplied by Optical Glass Limited are the reference materials to monitor fluorescence detection instrument performance and for standardization. Organic fluorescent dyes are dissolved in methylmethacrylate and the solution polymerized to produce a polymethylmethacrylate (PMMA) matrix, which provides a relatively stable environment for the enclosed organic fluorescent compounds. Polybead™ fluorescent polymer beads of Polysciences are used to calibrate the fluorescence spectrometer, flow pyrometer, plate reader etc. The organic fluorescent dyes are absorbed in the holes of polystyrene beads. Both the above two manufacturers don't mention calibration products for fluorescence microarray scanners. But in patent US2003/0105195, the inventor described the method of patterning the organic fluorescent dyes doped polymer matrix (PMMA, polyepoxide resin, polyamide) on the surface of a rigid slide to form a calibration slide for microarray scanners.
However, among these materials, the problem with photobleaching of fluorescent dyes still could not be resolved.
3. Broadband Fluorescence Emission Polymer as Calibration Material:
In the patents of US2003/0057379, US2003/0015668 and US6472671, a calibration tool for fluorescence microscopy is presented, which consists of a support of non-fluorescent quartz, a solid surface layer with a broadband fluorescence emitter, polyimide, and a thin opaque metal mask of non-fluorescent material. In a similar way, Clondiag Chip Technologies GmbH provides a novel array imaging standardization slide, which consists of an array of fluorescent spots with defined shape and intensity. In WO 02/077620, Clondiag patented manufacturing process guarantees stable fluorescence properties after ≧20 measurements. The fluorescent material is SU 8-10, a photo-sensitive broadband emitter polymer. Nevertheless, the polymer can also be photobleached under excitation, resulting in changes in the detected fluorescence intensity.
4. Organic Dye Doped Inorganic Solid Complex as Calibration Material:
US patent US2003/0015668 discloses a method to deposit an extremely thin layer of Cy3, Cy5 or other fluorescent dye doped glass by evaporation or sol-gel process on a non-fluorescent support and strengthened by baking at a relatively low temperature. But as the fluorescing material is again organic dye, simply encaged in inorganic matrix doesn't improve its photostability.
5. Inorganic Ion Doped Inorganic Solid Complex as Calibration Material:
Matech Co. provides fluorescent reference standards for 96 well plate readers. Each standard is made of a metal ion fluorophore doped in an inorganic glass host. The fluorescent ions used are Ce3+, U6+ and Eu3+. Comparison photobleaching data between the rare-earth doped glass, rhodamine B and fluorescein in PMMA have been taken. During weeks of exposure to high intensity excitation light, both organic dyes show significant photobleaching, whereas the rare-earth doped glass shows little. However, the manufactory method restricts its application in calibration with fine structures for microarray scanners.